HiPSC-derived 3D neural models reveal neurodevelopmental pathomechanisms of the Cockayne Syndrome B

Cockayne Syndrome B (CSB) is a hereditary multiorgan syndrome which—through largely unknown mechanisms—can affect the brain where it clinically presents with microcephaly, intellectual disability and demyelination. Using human induced pluripotent stem cell (hiPSC)-derived neural 3D models generated from CSB patient-derived and isogenic control lines, we here provide explanations for these three major neuropathological phenotypes. In our models, CSB deficiency is associated with (i) impaired cellular migration due to defective autophagy as an explanation for clinical microcephaly; (ii) altered neuronal network functionality and neurotransmitter GABA levels, which is suggestive of a disturbed GABA switch that likely impairs brain circuit formation and ultimately causes intellectual disability; and (iii) impaired oligodendrocyte maturation as a possible cause of the demyelination observed in children with CSB. Of note, the impaired migration and oligodendrocyte maturation could both be partially rescued by pharmacological HDAC inhibition. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00018-024-05406-w.


Introduction
The Cockayne Syndrome B (CSB) is a rare hereditary disease (prevalence ≈ 2.5 per million, [1]) with heterogeneous multi-organ defects including growth failure, retinal atrophy, deafness and a progeric skin phenotype.In addition, children with CSB develop severe neuropathological defects, with the cardinal phenotypes being microcephaly, intellectual disability and demyelination [2][3][4][5][6].Around 70% of all CS cases are caused by several mutations in the excision repair 6 chromatin remodeling factor (ERCC) 6 gene and manifest with varying neurological severity, the most severe being fatal during early childhood [7,8].Some cases of CS have been linked to ERCC8 (CSA protein) defects, usually resulting in the less severe forms of the disease [9,10].
CSB rodent models have proven very valuable for gaining insights into the clinical CSB phenotypes and the effects of CSB on the organism level [11][12][13][14][15], which lead to a good understanding of the mechanistic underpinning of the skin phenotype of CSB [16][17][18].However, the origin of the children's neurological defects is still enigmatic, because neurological defects cannot fully be modeled in rodents.Emerging in vitro approaches based on stem cells, e.g.human induced pluripotent stem cells (hiPSCs), can add to the current knowledge in human disease modeling and drug target identification, by providing excellent tools to investigate diseases and their underlying pathomechanisms [19][20][21][22][23][24][25].
CSB was originally found to be involved in the transcription-coupled nucleotide excision repair pathway (TC-NER) [26][27][28][29].However, the neuropathology of the patients cannot be well explained with this mechanism.Previous in vitro studies have therefore suggested a role of CSB in the brain, which is independent of its involvement in the TC-NER [30][31][32][33].Main findings include hindered neuronal differentiation and neuritogenesis, linked to reduced MAP2, as well as SYT9 and BDNF levels in 2D small hairpin (shRNA)-based CSB models of immortalized human neural progenitor cells (hNPC) and SH-SY5Y neuroblastoma cells [30,31].Vessoni et al. [32] found alterations in synapse density and reduced electrical activity in relation to a dysregulated Growth Hormone/Insulin-like Growth Factor-1 (GH/IGF-1) pathway in 2D iPSC-derived neuron/astrocyte mixed cultures and Liang et al. [33] identified Necdin as a CSB target, which promotes neuronal differentiation in 2D models of CS1AN human CSB fibroblasts and SH-SY5Y cells.Although these studies adumbrate the impact of CSB on brain development, we still lack the necessary understanding of why the clinical phenotypes arise and how we can treat them.Main limitations in current CSB models include non-physiological spatiotemporal microenvironments and low model complexity in 2D systems, missing cell types, such as oligodendrocytes, and a lack of appropriate control cell lines.These limitations outline the need for physiologically more relevant disease models, that better resemble the complexity of the developing human brain, and include isogenic controls.To investigate the brain phenotype of CSB in more physiologically relevant conditions, Szepanowski et al. [34] studied the effects of CSB on a transcriptomic level using 3D brain organoids derived from patient cells, providing valuable mechanistic information.
To add to this, we developed quality controlled CSB in vitro models, using patient derived and isogenic control cell lines, enabling the direct comparison between healthy and disease conditions and the identification of CSB-derived phenotypes.Study designs using multiple isogenic control pairs from different hiPSC donors have been shown to have an absolute power advantage of up to 60% compared to study designs without isogenic control pairs [35].Here, we use such a favorable two isogenic pair design to study different neurodevelopmental processes disrupted in CSB.We closed the current knowledge gap on CSB neuropathology, by utilizing two hiPSC-derived fit-for-purpose 3D cell culture models, one of which is patient-based, including human induced neural progenitor cell (hiNPC) neurospheres and 3D-differentiated BrainSpheres [36][37][38][39].The two different fit-for-purpose models were applied to answer different research questions concerning the underlying mechanisms of CSB.While hiNPC neurospheres enable the investigation of early developmental key events (KE) such as NPC proliferation, migration and initial terminal differentiation into neurons and astrocytes, modeling later KEs such as neural network formation and oligodendrogenesis benefit from more complex models such as 3D differentiated BrainSpheres [39].BrainSpheres have a complex 3D cytoarchitecture and consist of the relevant brain cell types, i.e. neurons of different subtypes, astrocytes and-facultatively-oligodendrocytes.Together, both in vitro models serve as ideal tools for investigating earlier and later neurodevelopmental processes and the underlying mechanisms of their disruption [36][37][38][39].

Cell lines
The commercial wild-type hiPSC-IMR90 line was obtained from WiCell (clone 4, Madison, USA).The patient-derived hiPSC-CS789 line was kindly provided by Prof. Egly from the IGBMC Strasbourg an has been sequenced at the IUF [40].ERCC6 CRISPR/Cas9 mutants CS789 Res and IMR90 KO were generated in house, as previously described [41].In brief, gRNAs (supplemental information (SI) Fig. S1) were designed using the CRISPR design tool CHOPCHOP (https:// chopc hop.cbu.uib.no/) and cloned into a modified version of the PX458 plasmid (Addgene #48138, Watertown, USA).The resulting bicistronic vector encoded the respective gRNA, Cas9 nuclease and GFP selection marker.gRNAs activity and efficiency were assessed via high resolution melt analysis (HRMA).HiPSCs cells were transfected with nuclease plasmids in antibiotic-free medium in a 6-well plate using Lipofectamine Stem (Thermo Fisher Scientific, Waltham, USA) or NEON electroporation system (Thermo Fisher Scientific, Waltham, USA).After 48 h, cells were sorted (FACS or MACS) and plated as single cells in a 96-well plate and duplicated after a week.Clones were lysed in proteinase K and genotyped by deep sequencing using a MiSeq Illumina (San Diego, CA) [41].Briefly, libraries were quantified using qBit4 (Thermo Fisher Scientific, Waltham, USA) and deep sequencing was performed according to the manufacturer's protocol (Illumina, San Diego, CA) at around 2000 reads per clone using custom made barcodes.Data were obtained in FASTQ format and analyzed using CRIS-PRnano.de[42].
To assure high and reliable cell culture quality, all hiPSC lines used in this study were quality controlled and banked, based on the recommendations of Tigges el al. [43].Briefly, cells were characterized via karyotyping, STR analysis, FACS analysis for pluripotency markers and viability, mycoplasma test and colony morphology.
2D neural inductions were performed with cell lines IMR90 WT and IMR90 KO , specifically for the generation of electrically active neural networks, which could not be achieved with the 3D induction protocol.The 2D inductions were performed according to Hartmann et al. [39].HiPSCcolonies were dissociated using the Gentle Cell Dissociation Reagent (#100-0485, Stemcell Technologies, Vancouver, Canada) and subsequently seeded with a cell density of 2 × 10 6 cells per well of a 6-well plate coated with polyethyleneimine (PEI, 0.1%; #181978, Sigma-Aldrich, Burlington, USA) and laminin (15 µg/ml; #LN521, Biolamina, Sweden), and cultivated in NIM medium supplemented with 10 µM ROCK inhibitor (only for the first 24 h after passaging; #HB2297, Hello Bio, Great Britain) under humidified conditions at 37 °C and 5% CO 2 .Cells were cultivated for 12 days, before medium was changed to neural progenitor medium (NPM), containing proliferation medium without hFGF, 20% (v/v) Knockout Serum Replacement (10828028, Invitrogen, Waltham, USA), 1:100 N2 supplement (17502-048, Invitrogen, Waltham, USA) and 20 ng/ml hFGF (#233-FB, R&D Systems, Minneapolis, USA).Medium was completely changed every second day.Cells were passaged on days 12 and 17 though enzymatic dissociation with Accutase (#07920, Stemcell Technologies, Canada) and transferred to a new PEI-laminin-coated 6-well plate.On day 21, hiNPCs were singularized with Accutase and frozen in neural progenitor medium containing 10% dimethyl sulfoxide (DMSO, #A994.1,Carl-Roth, Germany) and 10 µM ROCK inhibitor.Each thawn hiNPCs vial was diluted in 10 ml of the respective neural progenitor medium with 10 µM ROCK inhibitor (#HB2297, Hello Bio, Great Britain) and centrifuged at 300 × g for 5 min.The cell pellet was resuspended in 4 ml NPM medium with 10 µM ROCK inhibitor (#HB2297, Hello Bio, Great Britain) and transferred to one well of a 6-well plate (#83.3920,Sarstedt, Germany) coated with anti-adherence rinsing solution (#07010, Stemcell Technologies, Vancouver, Canada).Cells were cultivated in an orbital shaking incubator (#LT-X, Kuhner Shaker GmbH, Swiss) at 140 rpm, 12.5 mm diameter, 37 °C, 5% CO2, and 85% humidity for 7 days without feeding, to allow sphere formation.Medium was changed to NPC proliferation medium on day 7 for culture maintenance.Cells were fed every second day with NPC proliferation medium and mechanically passaged to 0.2 mm diameter when exceeding a size of ≈ 0.5 mm (McIlwain Tissue Chopper, Ted Pella).Spheres were maintained in proliferation medium for culture maintenance.
BrainSpheres differentiation was conducted according to the needs of the respective readout.Protocols are described in each section.

Proliferation
Human hiNPC spheres of 0.3 mm diameter were placed into separate wells of a Poly-HEMA-coated (#P3932, Merck, Darmstadt, Germany) U-bottom 96-well plate (Greiner, Austria) and statically cultured in proliferation medium for 3 days.The proliferation was assessed by measuring the increase in sphere size and by assessing the incorporation of BrdU into newly synthesized DNA.Spheres were imaged in brightfield mode (Thermo Fisher Scientific, Waltham, USA) from d0-to d3.The sphere size was automatically measured using the Cellomics ArrayScan Software and the slope of the size increase was calculated for each sphere.
The cell proliferation BrdU assay was performed on day 3 (#11669915001, Sigma Aldrich, Missouri, USA) according to the manufacturer's instructions.

Immunocytochemistry (ICC)
Samples were fixed with a final concentration of 4% paraformaldehyde (#P6148, Sigma Aldrich, Missouri, USA) for 30 min at 37 °C, followed by three PBS washing steps.The desired primary antibodies were diluted in 2% goat serum (G9023, Sigma Aldrich, Missouri, USA) and PBS-T (PBS in 0.1% (v/v) Triton X-100 (#T8787, Sigma Aldrich, Missouri, USA)).Subsequently, the antibody solution was added to the samples and incubated at 4 °C overnight.Samples were washed three times with PBS.Next, the secondary antibodies, or conjugated antibodies, were added to 1% Hoechst (33258, Sigma Aldrich, Missouri, USA) and 2% goat serum in PBS.The samples were incubated with the secondary antibody solution at 37 °C for 1 h.Finally, the samples were washed three times with PBS and imaged with the confocal laser scanning microscope TCS SP8 (Inverse DMi8CS, Leica Microsystems) or the Cellomics ArrayScan (Thermo Fisher Scientific, Waltham, USA).Floating spheres were positioned onto microscopy glass slides and covered with Aqua-Poly/Mount (#18606-20, Polysciences Inc., USA) and a cover glass before imaging.All antibodies are listed in the supplementary table (SI Table S3).
ICC image detection and quantification by high-content imaging analysis (HCA) was done using the Cellomics ArrayScan.Respective channels were automatically acquired with a 40 × objective magnification and a resolution of 552 × 552 pixel.30 randomly assigned fields of each 96-well were scanned.Automated image analysis was performed with the Thermo Scientific HCS Studio software, using the Colocalization analysis tool.Specifically, all O4 positive cells were detected and normalized to the total number of detected nuclei.

RNA sequencing
IMR90 WT and IMR90 KO lines were used for transcriptome analyses.The cell lines IMR90WT/IMR90KO were chosen for RNASeq analyses due to their clean, non-patient genetic CSB knock-out ensuring prohibition of a potential bias through the patient genetic background and allowing a clean transcriptome analysis based on a truncated, non-functional CSB protein.Here, 500 hiNPC spheres with a 0.1 mm diameter were plated onto poly-d-lysine (PDL, 0.1 mg/ mL, Merck, #P0899) and laminin (0.0125 mg/mL, Merck, #L2020)-coated 6-well plates and differentiated in CINDA medium for 3, 14 or 21 days.Total RNA was isolated using the RNeasy Mini Kit (#74104, Qiagen, Hilden, Germany) according to the manufacturer's instructions.RNA was sent to BGI Genomics Co., Ltd.(China) for RNA sequencing using the DNBseq platform and the reads were mapped to human reference genome hg38.Three biological replicates were performed for each cell line.
Library preparation: Total RNA sample quality control (QC) was done using the Agilent 2100 Bio analyzer (Agilent RNA 6000 Nano Kit).Subsequently, mRNA was purified using oligo (dT)-attached magnetic beads, and fragmented.After, synthesis of the first and second cDNA strands, end repair and "A" base was added to the 3′end.Adaptor ligands were added, and PCR was performed.PCR product purification was done with XP beads.QC was again done using the Agilent 2100 Bio analyzer.Double stranded PCR products were denatured and circularized by splint oligo sequencing.Resulting single strand circle DNA was formatted as the final library.The library was amplified with phi29 to make DNA nanoball (DNB).The DNBs were loaded into the patterned nanoarray and single end 50 (pair end 100/150) bases reads were generated in the way of combinatorial Probe Anchor Synthesis (cPAS).

Quantitative polymerase chain reaction (qPCR)
For KCC2 and NKCC1 expression, 60 hiNPC spheres of 0.1 mm diameter were plated onto poly-d-lysine (PDL, 0.1 mg/mL, Merck, #P0899) and laminin (0.0125 mg/mL, Merck, #L2020)-coated 48-well plates and differentiated in CINDA for 3, 14 or 21 days.For oligodendrocyte marker expression analyses, spheres were differentiated as described in the oligodendrocyte differentiation protocol, before harvesting.Total RNA was isolated using the RNeasy Mini Kit (#74104, Qiagen, Hilden, Germany) according to manufacturer's instructions.RNA was then reverse transcribed to cDNA (#205314, QuantiTec Reverse Transcription Kit, Qiagen, Hilden, Germany) according to the manufacturer's instructions and qPCR was performed (#204057, Quanti Fast SYBR Green Kit, Qiagen, Hilden, Germany) using the PCR-Cycler Rotor-Gene Q (Qiagen, Hilden, Germany).Primer sequences are listed in the SI Table S2.Because we did not perform primer efficiency testing, the results have to be handled with care.

Western blot (WB)
For CSB protein analyses, proliferating hiNPC spheres were analyzed.For all other markers, 750 hiNPC spheres with a diameter 0.1 mm diameter were plated into one well of a 6-well plate coated with poly-d-lysine (PDL, 0.1 mg/ mL, Merck, #P0899) and laminin (0.0125 mg/mL, Merck, #L2020).Spheres were cultivated under differentiation conditions in CINDA medium for 3 days.Cell pellets of each well were lysed in RIPA buffer (Cell Signaling Technology, Massachusetts, USA) and 1 mM protease inhibitor PMSF (Cell Signaling Technology) on ice for 30 min and centrifuged for 15 min at 4 °C at maximum speed.Supernatant of protein samples were separated by 10% SDS-polyacrylamide gel electrophoresis and blotted onto PVDF membrane (BioRAD, Hercules, USA).Blots were blocked in 5% BSA diluted in 0.1% TBS-Tween-20 (TBS-T) for 1 h at RT and subsequently incubated with antibodies of interest overnight at 4 °C according to the manufacturer's instructions.Blots were washed in TBS-T for 30 min and incubated with a 1:1000 dilution of HPR-conjugated secondary antibody (LI-COR Biosciences, Nebraska, USA) in 5% BSA in TBS-T at RT for 1 h.After final washing step of 30 min bands were visualized using ECL Prime (GE Healthcare, Freiburg, Germany) chemiluminescence substrate and the Odyssey imaging system (LI-COR Biosciences).Densitometry was carried out using Image Studio Lite software (LI-COR Biosciences).All antibodies are listed in the supplementary table (SI Table S3).All unprocessed blots can be found in supplementary figure S8.

Multielectrode arrays (MEA)
96-well cyto-view MEA plates (#M768-tMEA-96B, Axion Biosystems, Atlanta, USA) were coated with poly-l ornithine (PLO, 0.1 mg/ml, #P3655, Sigma Aldrich, Missouri, USA) and laminin (#LN521-05, 50 µg/ml, Biolamina, Sundbyberg, Sweden).3D induced hiNPCs of CS789 Res and CS789 cell lines were mechanically passaged to 0.1 mm diameter and transferred into CINDA+ differentiation medium, containing DMEM/F12 (31330038, Invitrogen, Waltham, USA), 1:50 B27 Plus supplement (A35828-01, Gibco, Billings, USA), 1:100 N2 supplement (17502-048, Invitrogen, Waltham, USA), 650 µg/ml creatin monohydrate (C3630, Sigma Aldrich, Burlington, USA), 100 U/ml Interferon-γ (300-02, Peprotech, Rock Hill USA), 20 ng/ml Neurotrophin-3 (450-03, Peprotech, Rock Hill USA), 20 µM Ascorbic acid (A5960, Sigma Aldrich, Burlington, USA), 1:100 (v/v) Penicillin/Streptomycin (P06-07100, PAN-Biotech, Aidenbach, Germany) and 300 µM d-cAMP (D0260, Sigma Aldrich, Burlington, USA).20 hiNPC spheres in 200 µl CINDA+ were plated per well and 12 wells were prepared per cell line.For the IMR90 WT and IMR90 KO lines, 3D induced BrainSpheres did not yield sufficient electrical activity.Therefore, an adapted 2D induction protocol was used.Here, one proliferating sphere of approx.0.2-0.3mm size was plated in 200 µl CINDA+ per well and 12 wells were prepared per cell line.All lines were subsequently cultivated at 37 °C in a humidified atmosphere of 5% CO 2 for up to 7 weeks.Cells were fed every two to three days, by removing 100 µl and adding 100 µl CINDA + medium.The electrical activity was measured for 15 min every week after an equilibration time of 15 min on the Maestro Pro MEA system (Axion Biosystems, Atlanta USA).During the time of the measurement, temperature and CO 2 were kept stable and equivalent to the cultivation conditions.Data recording was operated by the Axion Integrated Studios (AxIS) navigator software (version 3.1.2,Axion Biosystems, Atlanta, USA) with a sampling frequency of 12.5 kHz and a digital band-pass filter of 200-3000 Hz.Subsequent spike detection was performed using the method "adaptive threshold crossing" with a threshold of 6 root mean square (rms) noise on each electrode and a pre-and post-spike duration of 0.84 ms and 2.16 ms, respectively.An electrode was termed "active" with at least 5 spikes per min.Quantification of general electrical activity and neuronal network activity was performed with the Neural Metric Tool software (version 3.1.7,Axion Biosystems, Atlanta, USA).For burst detection, the method "Inter-spike interval (ISI) threshold" was used with a minimum of 5 contributing spikes and a maximum ISI of 100 ms.Network bursts were identified using the algorithm "envelope" with a threshold factor of 1.5, a minimal inter-burst interval (IBI) of 100 ms, at least 35% participating electrodes, and 75% burst inclusion.Parameters for neuronal activity (percentage of active electrodes and number of spikes) as well as for network maturation and synchronicity (number of network bursts, number of spikes per network bursts and area under normalized cross correlation) were analyzed.

GC-mass spectrometry
For GC-MS analyses, 500 hiNPC spheres with a diameter of 0.1 mm were plated into one well of a 6-well plate coated with poly-d-lysine (PDL, 0.1 mg/ml, Merck, #P0899) and laminin (0.0125 mg/ml, Merck, #L2020).Spheres were cultivated under differentiation conditions in CINDA medium for 14 days.As a wash control, 1 mM Tricarballylic acid (T53503, Sigma Aldrich, Missouri, USA) was added to each well of the cell culture medium, immediately before harvesting on day 14.Cells in each well were washed four times with ice cold 0.9% (w/v) saline in MilliQ water, before being collected in 2 ml of 0.9% (w/v) saline (3957.1,Roth, Karlsruhe, Germany) in MilliQ water. 2 ml methanol (N41.1,Roth, Karlsruhe, Germany) were supplemented with 250 µl internal standard (ISTD; 10 µM final; ribitol; A5502-5G, Sigma Aldrich, Missouri, USA). 2 ml methanol-ISTD solution was added to 2 ml of cell suspension and samples were shock frozen in liquid nitrogen.Upon thawing on ice, 1 ml chloroform (3313.1 Roth, Karlsruhe, Germany) was added to 4 ml sample solution and the mixture was incubated on ice and frequently vortexed for 10 min, before resting for 5 min on ice.The samples were centrifuged for 10 min at 4 °C and 4000 × g.Subsequently, the aqueous phase (top layer) was collected in a separate tube and the remaining organic phase was washed with 2 ml ice-cold MilliQ water.After another centrifugation cycle at 4 °C and 4000 × g for 10 min, the aqueous phase was collected and pooled with the first collection tube.The sample was filled with 11.5 ml MilliQ water, to reduce the amount of the organic solved < 15%.Samples were frozen for at − 80 °C, before lyophilization was carried out.Dried samples were resuspended in 500 µl MilliQ water, of which 20 µl were mixed with 50 µl methanol and dried via vacuum centrifugation for derivatization and GC-MS measurement.
The polar metabolites were derivatized for GC-MS analysis according to the method described by Gu et al. [47].The derivatization process was executed using an MPS-Dualhead autosampler (Gerstel, Mülheim an der Ruhr, Germany).First, 10 µl of methoxyamine hydrochloride (10440364, Thermo Fisher Scientific, Fisher Scientific Chemicals, Waltham, Massachusetts, USA; freshly prepared at 20 mg/ ml in pure pyridine from) were added, and the samples were shaken at 37 °C for 90 min.Next, 90 µl of N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA; Macherey-Nagel, Düren, Germany) were added and shaken at 37 °C for 30 min, followed by a 2-h incubation at room temperature.Metabolite analysis was conducted using a 7890B gas chromatography system connected to a 7200 QTOF mass spectrometer from Agilent Technologies, as previously described by Shim et al. [48].Compound identification was performed using MassHunter Qualitative software (v b08, Agilent Technologies, Santa Clara, USA) by comparing the mass spectra to an in-house library of authentic standards and the NIST14 Mass Spectral Library (available at https:// www.nist.gov/ srd/ nist-stand ard-refer ence-datab ase-1a-v14).Peak areas were integrated using MassHunter Quantitative software (v b08, Agilent Technologies, Santa Clara, USA) and normalized to the internal standard ribitol (Sigma Aldrich, Missouri, USA).

Migration, proliferation, western blot and mass spectrometry analyses
GraphPad Prism was used to create visual graphs and analyses.Statistical significance was determined by unpaired twotailed t-tests.Migration rescue experiments were analyzed using ANOVA with the Dunnett test for multiple comparisons.A p value below 0.05 was termed significant.Three biological replicates were performed for each cell line and each endpoint (three to four for WB).Migration and proliferation (including cytotoxicity and viability) additionally included three to ten technical replicates.Each biological replicate stems from a distinct neural differentiation.
Fig. 1 Human iPSC-derived cell models to study adversities in neurodevelopmental key events linked to Cockayne Syndrome B (CSB).A The two hiPSC-based CSB disease models used in this study include the commercially available IMR90 WT cell line and its CSBdeficient control line IMR90 KO , as well as the patient-derived cell line CS789 and its isogenic control CS789 Res , with partially restored CSB expression.Both IMR90 KO and CS789 Res were produced via CRISPR Cas (see also Supplemental Information Fig. S1).All hiPSC lines were quality controlled and cell banks were prepared according to Tigges et al. [43].B Illustration depicting different fit-for-purpose models.Human iPSCs were neurally induced to hiNPC neurospheres, which were used to assess NPC proliferation after.HiNPCs were plated onto coated surfaces for subsequent differentiation, to assess NPC migration and differentiated into neurons and astrocytes after 3 DIV (middle).In order to obtain electrically active neural networks, hiNPC neurospheres were plated onto coated MEA plates for subsequent differentiation and electrical activity measurement over 7 WIV (left).The differentiation of oligodendrocytes requires a long-term differentiation protocol.HiNPC neurospheres were differentiated in a shaking incubator for 8 WIV to obtain BrainSpheres, which consist of neurons, astrocytes and oligodendrocytes (right).C Immunoblotting of CSB levels in neurospheres of the four hiNPC lines, normalized to the loading control HSP90 and to the respective control (left) or IMR90 WT (right).Representative images are depicted and graphs visualize differences in protein content due to mutations in the CSB gene or between CSB expressing lines, respectively.Graphs depict N = 3 biological replicates, mean ± SEM.Statistical analyses were performed using unpaired two-tailed t-tests.A p-value below 0.05 was termed significant.D HiNPC spheres were plated, differentiated for 3DIV and subsequently stained for nuclei (Hoechst, blue), neurons (MAP2, yellow) and astrocytes (AQP4, magenta).E Transcriptome analyses were performed by plating hiNPC neurospheres onto a coated surface, and differentiating these for 3,

RNA seq
DEG criteria was set to |log2|≥ 1 and Qvalue of ≤ 0.05 were used for subsequent analyses and KEGG pathway mapping.Individually analyzed genes had a minimum FPKM (Fragments Per Kilobase per Million mapped fragments) of 1. Analyses and visualization of gene expression parameters were done using the Dr. Tom software (BGI Genomics Co., Ltd.).Three biological replicates were performed for each cell line.Each biological replicate stems from a distinct neural differentiation.

MEAs
Statistical analyses of MEA results were performed in GraphPad using Mixed-effects analysis with the Sidak test for multiple comparisons (N = 8-12 wells with n = 64-96 electrodes).Cells of one neural induction were used for all wells.A p value below 0.05 was termed significant.

Materials and correspondence
Requests for further information should be directed to and will be fulfilled by the corresponding author, Dr. Katharina Koch (Katharina.Koch@IUF-Duesseldorf.de).

Two hiPSC models for neurodevelopmental key event analyses in the cockayne syndrome B
For investigating the CSB neuropathology, we established two distinct hiPSC disease models, each including a CSB-deficient and a CSB-expressing cell line.The first model consists of the commercially available hiPSC line IMR90 WT , which expresses the CSB wildtype in a healthy donor genetic background and an IMR90 KO line generated via CRISPR Cas technology, by introducing a 13 bp deletion into exon 5 of the ERCC6 gene (Fig. 1A, left; supplemental information (SI) Fig. S1).The second model consists of the patient-derived hiPSC line CS789 and the isogenic control line CS789 Res .The patient cell line holds a point mutation (2047C > T) in exon 10 of the ERCC6 gene, which leads to a premature stop codon.The genetic rescue of the control line (CS789 Res ) was generated via CRISPR Cas technology, through a 9 bp in-frame deletion, which removes the stop codon (Fig. 1A, right; SI Fig. S1).The donor of the patient hiPSCs was classified with Cerebro-oculofacio-skeletal syndrome (COFS), one of the most severe forms of CSB (supplemental information (SI) Table S1).The patient was, amongst others, afflicted with congenital microcephaly, severe intellectual disability and seizures.He passed away at 10 months of age.
The hiPSC lines were neurally induced into neurospheres containing hiNPCs, and subsequently either differentiated on an artificial extracellular matrix or floating as 3D BrainSpheres into neurons, astrocytes and facultatively oligodendrocytes (Fig. 1B), thereby generating different fit-for-purpose models.
First, we confirmed by western blot, that the CSB protein is expressed in hiNPC neurospheres of both healthy cell lines (IMR90 WT and CS789 Res ), but not in either of the disease cell lines IMR90 KO and CS789 (Fig. 1C left).Despite varying levels of expression (Fig. 1C right), the CSB protein was detected in both IMR90 and CS789 Res lysates.Due to the non-isogenic nature of the two lines, such differences in expression levels of the CSB protein can be expected.We next confirmed that hiNPC neurospheres of all four cell lines are able to differentiate into cells of the neural lineage, containing both neurons and astrocytes (Fig. 1D).
To obtain an understanding of the consequences of CSBdeficiency for the hiNPCs' bulk transcriptome, we performed mRNA Sequencing (RNAseq) analyses.Here we decided for the control IMR90 WT and the IMR90 KO lines, in order to understand the effects of the clean CSB mutation and to exclude impacts of the patient specific genetic background.Transcriptome analyses were performed by plating hiNPC neurospheres onto a coated surface, and differentiating these for 3, 14 and 21 days in vitro (DIV).In total, 893 genes were differentially expressed in IMR90 KO compared to IMR90 WT cells across all time points (Fig. 1E).Only significantly regulated genes with a |log2| of ≥ 1 were analyzed.These differentially expressed genes (DEG) were mapped to Kegg pathways and the top regulated pathways are depicted in Fig. 1F.Most DEGs map to autophagy (ID4140) and mitophagy (ID4137), as well as associated pathways such as MAPK (ID4010), RAP1 (ID4015), PI3K (ID4151) and Ras (ID4014) signaling.Another high number of DEGs map Fig. 2 CSB-deficiency is associated with inhibited neural progenitor cell migration and alterations in focal adhesion and autophagy.A 0.3 mm hiNPC neurospheres were plated and differentiated for 3 DIV.Cell migration was assessed by measuring both migration distance of furthest migrated cells (bars) and the total number of migrated nuclei (dots).Exemplary brightfield images are shown on the right.The graph depicts N = 3 biological replicates with n = 5-10 spheres each, mean ± SEM.B The heat map shows selected significantly regulated DEGs of focal adhesion-related pathways, identified using RNA Sequencing in the IMR90 WT and IMR90 KO cell lines.DEG criteria: |log2| > = 1 and Qvalue of < = 0.05.C Representative stainings of the migration area of plated hiNPC neurospheres after 3 DIV, showing nuclei (blue) and the cytoskeletal marker F-actin (white).D Heatmap of the 140 differentially regulated autophagy-associated genes of IMR90 KO compared to IMR90 WT acoss 3, 14 and 21 DIV as identified using RNA Sequencing (right), and separately highlighted DEGs of interest (left).DEG criteria: |log2| > = 1 and Qvalue of < = 0.05.E Representative ICC images of differentiating hiNPC neurospheres after 3DIV, showing nuclei (blue) and lysosomes (LAMP2, magenta).F Western Blot of plated and 3DIV differentiated hiNPC spheres, quantifying the fold change in LC3A, LAMP2 and mTOR protein levels, normalized to the loading control HSP90 and the respective CSB expressing control cell line.Graphs depict N = 3 biological replicates, mean ± SEM.For A and F a p-value below 0.05 was termed significant.Statistical analyses were performed using unpaired twotailed t-tests.Abbreviations: NPCs neural progenitor cells, DEG differentially expressed gene, DIV days in vitro, ICC Immunocytochemistry ◂ to Cell adhesion molecules (CAMs, ID4514), the regulation of the actin cytoskeleton (ID4810) and focal adhesions (ID4510), as well as calcium signaling (ID4020) and glutamatergic synapses (ID4724).

CSB-deficiency is associated with inhibited neural progenitor cell migration and alterations in focal adhesion and autophagy
Microcephaly is a major neuropathological finding in CSB patients [1,7].Two underlying cellular deficiencies that might cause microcephaly are inhibited NPC proliferation or migration [49,50].Therefore, we investigated these neurodevelopmental KEs in the CSB proficient and deficient in vitro models.Proliferation was assessed in hiNPC neurospheres, by measuring the sphere diameter over time, and by quantifying the incorporation of Bromodeoxyuridine (BrdU) into newly synthetized DNA.No significant differences between healthy and disease neurospheres were observed in either proliferation assay (SI Fig. S2).Next, we assessed the migration capacity of plated hiNPC neurospheres by measuring total migration distance from the rim of the sphere core to the furthest cell that migrated out of the sphere core, and by counting the total number of migrated nuclei after 3 DIV within the migration area.We observed a significant reduction of the migration distance (25-35%), and a significantly decreased number of migrated nuclei (25-60%) within the migration area using hiNPCs from disease cell lines, compared to their respective controls (Fig. 2A).In support of the inhibited migration, transciptome analyses present significant upregulation of the focal adhesionspecfic gene expression markers Integrin ß1 (ITGB1), Integrin ß4 (ITGB4), Talin-1 (TLN1) and Vinculin (VCL) in the IMR90 KO , compard to the healthy IMR90 WT hiNPC neurospheres after 3, 14 and 21 DIV (Fig. 2B).An important intracellulart part of focal adhesions during migration are actin stress fibers.In line with the altered expression of genes involved in regulating the actin cytoskeleton (Fig. 1G), we observed thickened and elongated actin stress fibers in the migration area of plated disease hiNPC neurospheres after 3DIV (Fig. 2C).Inhibited focal adhesion turnover and subsequent migration inhibition thus seems to be one in vitro feature of CSB-deficiency.
In addition to the actin-and focal adhesion-related genes, 141 autophagy-associated mRNAs are significantly regulated in the disease IMR90 KO , compared to IMR90 WT differentiated neurospheres (Fig. 2D, right).Gene expression of the autophagosome markers microtubule-associated proteins 1A/1B light chain 3A (LC3A) and 3C (LC3C) are highly upregulated in the disease differentiated neurospheres.Additionally, the lysosome marker lysosomal-associated membrane protein-2 (LAMP2) and p62, a gene coding for a protein that taggs intracellular material for targeted autophagy, are also upregulated in the disease differentiated neurospheres (Fig. 2D, left).On the protein level, we identified LAMP2 accumulation in immunocytochemical stainings (ICC, Fig. 2E) and confirmed significant LC3A and LAMP2 accumulation in both CSB-deficient IMR90 KO and CS789 lines by Western Blot (WB) analyses of plated hiNPC neurospheres after 3 DIV (Fig. 2F).The accumulation of these proteins is a general marker for defective autophagy.Futhermore, a decreased level of mammalian target of rapamycin (mTOR), an autophagy-inhibitor, in IMR90 KO hints towards an increased demand for autophagy in the disease cells (Fig. 2F).

The HDAC6-inhibitor Tubastatin A partially rescues the migration phenotype of disease hiNPC neurospheres
A previous study on CSB-deficient fibroblasts suggested CSB to interact with α-tubulin acetyltransferase MEC-17 and histone deacetylase 6 (HDAC6).MEC-17 acetylates α-tubulin, thereby supporting cargo transport and autophagosome with lysosome fusion, while HDAC6 mediates histone deacetylation [18].We observed significantly decreased acetyl-α-tubulin levels and increased HDAC6 levels in differentiated and plated disease hiNPC neurospheres after 3DIV, compared to their respective healthy controls also after 3DIV (Fig. 3A).Low levels of α-tubulin acetylation generally suggest inhibited autophagy in the cell.
Since targeted autophagy is an important facilitator of focal adhesion turnover, which in turn enables cell Fig. 3 HDAC6-inhibitor Tubastatin A partially rescues the adverse migration phenotype of CSB-deficient cells.A Exemplary images and quantifications of Western Blot analyses depicting the fold change in acetyl-alpha-tubulin and HDAC6 protein levels, normalized to the loading control HSP90 and the respective control cell line.Graphs depict N = 3 biological replicates, mean ± SEM.B 0.3 mm hiNPC neurospheres were plated and differentiated under exposure to TubA (left) or CQ (right), before the migration distance was measured on DIV 3. The migration distance of both healthy and disease cell lines was normalized to the solvent control (SC) of the respective healthy cell line within each graph.Viability and cytotoxicity were assessed in parallel (Supplemental Information Figure S3).Graphs depict N = 3 biological replicates with n = 5-10 spheres each.C Illustration of the presumed CSB mechanism: CSB-deficiency correlates with increased HDAC6 expression (1) and reduced a-tubulin acetylation.This causes defective autophagy, specifically impaired autophagosome and lysosome fusion (2) and degradation ( 3) and hence lysosome accumulation.This in turn inhibits migration through inefficient focal adhesion turnover and altered regulation of the actin cytoskeleton (4).This cellular disease phenotype can be partially rescued by Tubastatin A and Chloroquine exposure of CSB-deficient cells.For A and B, a p-value below 0.05 was termed significant.Statistical analyses in A were performed using unpaired two-tailed t-tests and in B using one-way ANOVA with post-hoc Dunnett tests.Abbreviations: NPCs neural progenitor cells, TubA Tubastatin A, CQ Chloroquine.Figure created with biorender.com◂ migration, we performed HDAC-inhibition experiments to link HDAC activity to altered migration of the CSB disease models.Therefore, we treated the migrating hiNPC spheres at the time of plating with the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) or the HDAC-6-specific inhibitor Tubastatin A (TubA) for 3 DIV.SAHA did not significantly rescue the migration of the CSB-deficient cell lines, yet reduced the cell viability significantly even at low concentrations (SI Fig. S3).Treatment with TubA, however, successfully rescued the reduced migration in both IMR90 KO and CS789 disease lines, at noncytotoxic concentrations (Fig. 3B left, see also SI Fig. S3).To further strengthen the causal link between autophagy and migration, we inhibited the autophagy in migrating cells with chloroquine (CQ), a suppressor of autophagosome and lysosome fusion.Our results show a significant migration inhibition at non-cytotoxic CQ concentrations in both healthy cell lines (Fig. 3B right, SI Fig. S3).Since autophagy is already compromised in the disease cell lines, CQ does not further reduce migration of IMR90 KO or CS789 at non-cytotoxic concentrations.Considering these results, we propose a putative CSB mechanism: CSBdeficiency correlates with increased HDAC6 expression and reduced a-tubulin acetylation.This causes defective autophagy, specifically impaired autophagosome-lysosome fusion and degradation and thus lysosome accumulation.This in turn inhibits migration through inefficient focal adhesion turnover and altered regulation of the actin cytoskeleton (Fig. 3C).
To get a functional readout of the neural network formation, we recorded the electrical activity of neural networks derived from all cell models on microelectrode arrays (MEA).HiNPC neurospheres were plated onto 96-well MEAs and differentiated for neural network formation over a period of 7 weeks.This allows us to follow the formation of neural networks over time.Using MEAs, we can analyze multiple parameters, that are spike-burst-and network-related and represent single neuronal activity, single neuronal and network maturation, respectively (Fig. 4C) [52].The number of active electrodes increased in all neural networks and plateaued at approx.4-5 active electrodes per well (Fig. 4B).Representative spike raster plots (SRP) are shown in Fig. 4D.The SPR of each cell line depicts the spikes (black) and bursts (blue) of the first 30 s of the 15 min measurement after 6 weeks in vitro (WIV).We observed differences in some of the measured MEA parameters, between CSB-deficient and -proficient networks, i.e. number of spikes, number of network bursts, area under normalized cross correlation (network synchrony) and number of spikes per network burst (Fig. 4E).The IMR90 KO networks show no significant difference in the number of spikes compared to the IMR90 WT networks.However, a significant reduction of network bursts and a significant increase in area under normalized cross correlation and number of spikes per network bursts can be seen, starting from 5 WIV.Similarly, CS789 shows significantly increased area under normalized cross correlation and number of spikes per network bursts.However, the parameters 'number of network bursts' and 'number of spikes' are both significantly increased in CS789 compared to its control CS789 Res .Although the two different models cannot be directly compared, alterations on neural networks are similar, both indicating an increased electrical activity of the disease cell lines compared to their healthy controls.We observed interesting differences regarding the number of network bursts between the two genetic backgrounds in the IMR90WT/IMR90KO and CS789/ CS789Res systems that need to be investigated in follow up studies.The significant changes in electrical activity of both model systems hint towards an altered neural network formation in the disease system and hence are in line with Fig. 4 Microelectrode Array measurements reveal altered neural network activity in CSB-deficient networks.A Heatmap of the 36 differentially regulated synapse-associated genes of IMR90 KO compared to IMR90 WT accoss 3, 14 and 21 DIV.DEG criteria: |log2|> = 1 and Qvalue of < = 0.05.B HiNPC neurospheres were plated onto MEA plates (96-well, 8 electrodes per well) and differentiated for neural network formation.The neural network development was assessed by measuring the electrical signals over 7 weeks.The graphs depict the number of active electrodes normalized to the number of wells over time (N = 8-12 wells with n = 64-96 electrodes each; mean ± SEM).C Illustration of the MEA-derived parameters single spikes, bursts, network bursts and network synchronicity.D Representative spike raster plots (SRP) of one well of each cell line after 6 WIV.Displayed are the spikes (black) and bursts (blue) of the first 30 s of the 15 min total measurement.E The electrical activity of neural networks over time is depicted and compared to the respective control.The graphs show the mean ± SEM of three biological replicates for the selected parameters number of spikes, number of network bursts, area under normalized cross correlation (network synchronicity) and number of spikes per network burst (per replicate 8-12 wells with 64-96 electrodes were evaluated).Each time point comprises a 15 min measurement.A p-value below 0.05 was termed significant.Statistical analyses of MEA results were performed using Mixed-effects analysis with the Sidak test for multiple comparisons.Abbreviations: NPCs neural progenitor cells, MEA microelectrode array.Figure created with biorender.com◂ the transcriptome results.Noteworthy are especially the alterations of IMR90 KO and CS789 in the network activity, compared to their respective controls.Rescue trials with above mentioned HDAC-inhibitors TubA and SAHA did not change the neural network activities of either network (SI Fig. S5).

Altered GABA levels and KCC2 expression hint towards a delayed GABA switch in disease cell lines
In order to evaluate, whether the increase of spiking in CSB-deficient neural models is caused by excitatory neurotransmitter accumulation, we performed Mass Spectrometry (MS) analyses.For that hiNPC neurospheres were plated and differentiated for 14 DIV.Excitatory glutamate levels and GABA levels, show no significant difference between disease and control neurospheres (SI Fig. S6 and the supplementary SI Table S4).However, their respective GABA/ glutamate ratios, calculated from the relative metabolite concentrations, show an accumulation of GABA in both CSB-deficient systems, with significant accumulation in CS789 compared to CS789 Res (Fig. 5A).The neurotransmitter GABA has excitatory consequences before the postnatal GABA-switch, and holds inhibitory functions thereafter.The transition of this GABA switch is initiated by crucial changes in gene expression of the chloride importer Na-K-2Cl cotransporter isoform 1 (NKCC1) and the exporter K-Cl cotransporter isoform 2 (KCC2).During the GABA-switch, NKCC1 expression decreases, while KCC2 expression increases.As active chloride transporters, they are responsible for the high and low concentrations of intracellular chlorine before and after GABA switch, respectively [53].The control IMR90 WT -derived neural networks mimic this development over 21 DIV with increasing NKCC1 and decreasing KCC2 expression.In contrast, KCC2 is not upregulated in the CSB-deficient networks derived from IMR90 KO , indicating a disturbed GABA switch (analyzed via RNA Sequencing, Fig. 5B left).A similar picture is

CSB-deficiency leads to hindered oligodendrocyte maturation
Demyelination is a common neuropathological phenotype of CSB patients [1,2,7,14,51].Generating hiPSC-based BrainSpheres containing oligodendrocytes in addition to neurons and astrocytes in 3D requires a long-term protocol, that cultures the floating spheres in a shaking incubator (Fig. 1B).Here, BrainSpheres provide a more mature model compared to hiNPC neurospheres, due to their longer maturation period in 3D.After 8 weeks, Brain-Spheres were analyzed by immunocytochemical staining for neurons (β-III-tubulin) and oligodendrocytes (O4; Fig. 6A).No obvious morphological or numerical differences were observed in O4-stained oligodendrocytes differentiated from disease and respective control hiPSCs.In addition, hiNPC neurospheres were plated for subsequent adherent oligodendrocyte differentiation.After 4 weeks, cells were stained for the pan-oligodendrocyte marker O4 and scanned with a high-content imaging platform.Scanned images were then analyzed for the number of O4 positive cells, using a colocalization tool, which colocalizes nuclei and O4 staining in ICC images (Fig. 6B and SI Fig. S7).No significant differences in O4 staining was detected.Myelin formation is dependent on oligodendrocyte differentiation and their maturation.Therefore, we explored if CSB-deficiency impacts oligodendrocyte maturation, by analyzing the gene expressions of panand maturation stage-specific oligodendrocyte markers (Fig. 6C).No significant difference in the expression of the pan-oligodendrocyte marker SOX10 between CSB-proficient and -deficient BrainSpheres was observed.A second pan-oligodendrocyte marker, Olig2, was expressed significantly less in BrainSpheres from IMR90 KO , compared to IMR90 WT , yet this was not seen in CS789 compared to CS789 Res .The oligodendrocyte progenitor cell (OPC) marker FABP7 was overexpressed in both, IMR90 KO (significantly) and CS789-derived BrainSpheres.The OPC and pre-oligodendrocyte (pre-OL) markers NG2 and PDG-FRa were both underexpressed in IMR90 KO , while CS789 underexpressed PDGFRa, but not NG2.Correspondingly, expression of the immature and myelinating OL markers CNPase and MBP were underrepresented in both CSBdeficient BrainSpheres, compared to their respective controls.PLP was not differentially expressed in either cell system.These results point to a delayed maturation of developing oligodendrocytes in the CSB-deficient models, with an overexpression of the OPC-specific gene FABP7 and an underexpression of genes specific for more mature oligodendrocyte stages.
In the next step we assessed, whether this impaired oligodendrocyte maturation is mediated by HDACs, similar to the CSB consequences on migration.Between 6 to 8 WIV, a time where oligodendrocyte maturation takes place in Brain-Spheres, IMR90 KO and CS789 BrainSpheres were treated with 740 nM TubA or 50 nM SAHA (Fig. 6D).Both HDAC inhibitors significantly antagonized the CSB deficiency-dependent underexpression of the pre-OL marker PDGFRa in IMR90 KO and CS789.SAHA additionally induces a significant increase in the expression of the myelinating OL marker MBP in both CSB-deficient BrainSpheres (Fig. 6E).

Discussion
Mimicking human disease and identifying treatments with animal models often undermines expectations.Especially for diseases involving the brain, translation from animals to humans is challenging.Species differences in brain physiology and kinetic properties are key here, with high dropout rates in drug development pointing to this [54].Drugs developed for CNS diseases display the second highest attrition rates right after cancer drugs with causes of drug failure allocating to lack of efficacy and second most frequently to toxicity [55].As an example, drug development for treating Alzheimer's disease alone produced over 99% failure rates [56].Similarly, treatments for neurodevelopmental disorders like autism spectrum disorders [57] are sparse.This is mainly due to the lack of pathophysiological understanding of the disease and a consecutive lack of known drug targets.In this study we aim at setting an example for unraveling molecular and cellular causes of a severe neurodevelopmental disease, the Cockayne Syndrome B (CSB), using 3D neural models like hiPSC-derived neurospheres and Brain-Spheres.We identified in vitro phenotypes that we relate to the children's pathophysiology and based on that propose novel treatment strategies for this devastating disease.
CSB is a heterogeneous hereditary disease with a spectrum of clinical phenotypes highly depending on the associated mutant genotype.However, common pathophysiological brain features of CSB patients include microcephaly, intellectual disability and demyelination [1,2,7,14].In this work, we provide for the first time mechanistic explanations for the cardinal brain phenotypes observed in CSB patients.We here use two 3D hiPSC-derived neural CSB models and their isogenic controls, a CSB patient-derived line and a genome-edited healthy donor hiPSC line carrying a truncating CSB mutation, both of which result in CSB protein deficiency.Specifically, our results suggest that CSB deficiency inhibits migration through defective autophagy, which is consistent with the clinical microcephaly observed in CSB patients.Further observations of altered electrophysiology and changes in GABA neurotransmitter levels in CSB-deficient neural networks indicate that a disturbed GABA switch is involved in altered brain circuit formation, ultimately leading to intellectual disability in patients.In addition, the impaired oligodendrocyte maturation we observed in CSB-deficient BrainSpheres provides an explanation for the demyelination observed in children with CSB.Therefore, using human-based 3D in vitro models, we identified multiple cellular pathomechanisms of CSB deficiency and were able to link them to the three cardinal brain phenotypes of CSB patients.
Microcephaly can be caused by dysfunctional NPC proliferation, migration, neuronal differentiation or apoptosis [49,50].We are the first to show that deficiency of the CSB protein causes NPC migration defects.Others previously reported on disrupted neuronal differentiation and neurite outgrowth as a consequence of CSB-deficiency in 2D immortalized NPC and hiPSC-derived neurons, respectively [30,31], which might also contribute to the microcephalic CSB phenotype.We did not observe an impairment of neuronal differentiation in the differentiating CSB-deficient neurospheres (SI Fig. S4), which could be explained by the large heterogeneity of CSB phenotypes between patients.Yet, these differences might also arise due to the different cell systems (immortalized NPCs vs. hiNPCs) or dimensionality (2D vs. 3D).Next, we substantiated the findings of impaired migration by mechanistic understanding.The disrupted migration of CSB-deficient hiNPC neurospheres is accompanied by altered markers of autophagy, i.e. dysregulated auto-and mitophagy-related gene and protein expressions and a reduced amount of acetylated α-tubulin.Disrupted autophagy based on malfunctioning HDACs was recently established as a major mechanism in the skin pathology of CSB patients [18].Here we extend this mechanism from the skin to the developing brain, by showing that the HDAC6specific inhibitor Tubastatin A rescues the inhibited migration in the CSB-deficient neurospheres.This leads us to hypothesize that HDAC-dependent defective autophagy is the cause for the impaired migration.This hypothesis is supported by the well-established knowledge that targeted autophagy plays a major role in focal adhesion turnover, which in turn facilitates cell migration (Fig. 3C) [58][59][60].HDAC-inhibitors can have highly context-dependent offtarget effects, such as decreased proliferation, increased cell death or histone modifications, which need to be evaluated individually, ideally in an organism-specific manner.However, selective HDAC-inhibitors are expected to have a high molecular specificity [61].
Besides migration, we also studied the functionalities of developing CSB-proficient and -deficient neural networks over 7 WIV on MEAs, as they provide a promising tool to investigate disease-associated alterations in neural circuit formation in vitro [62,63].Brain circuit formation is precisely orchestrated during brain development and a disruption leads to numerous pathological defects, many of which culminate in intellectual disabilities [64][65][66].Mutations in the CSB spectrum are also frequently accompanied by limited cognitive function and delayed neurodevelopment [1,2,7], leading us to speculate that disrupted circuit formation might be one of the underlying causes.Both CSB-deficient in vitro neural networks show increased and/or accelerated electrophysiological parameters compared to their healthy controls over time.The differences seen in some parameters when cross-comparing the two models may arise from the individual properties of the distinct cell lines, which underlines the necessity of isogenic controls for such disease models.In addition, neurotransmitter analyses coupled with transcriptional profiling identified elevated GABA levels, and a simultaneous delay in KCC2 expression, as the possible underlying reason for the increased electrophysiological activities on MEAs.GABA is a fundamentally important neurotransmitter with prenatal excitatory functions and a postnatal shift towards inhibition.This shift is realized by transcriptional up-regulation of the K + /Cl − co-transporter KCC2, which actively lowers intracellular chlorine levels and thereby reverses the passive Cl − transport by the GABA receptor [67].A disruption of this pivotal shift has so far not been described for CSB patients, however, it causes developmental delays and disorders in other neurodevelopmental diseases, such as autism spectrum disorder or attention deficit hyperactivity disorder (ADHD) [67,68].Opposite electrophysiological findings in hiPSC-derived neuron/ Fig. 6 CSB-deficiency leads to hindered oligodendrocyte maturation.HiNPC neurospheres were pre-differentiated in designated oligodendrocyte differentiation medium for 8 weeks in an orbital shaking incubator to generate oligodendrocyte-containing BrainSpheres.A Representative 3D BrainSphere ICC stainings after 8 WIV, showing nuclei (Hoechst, blue), neurons (β-III tubulin, magenta) and oligodendrocytes (O4, cyan).B Quantification of O4 positive cells after 4 weeks of adherent oligodendrocyte differentiation, normalized to the respective healthy control cell line.C Analyses of oligodendrocyte marker mRNA expression via qPCR after 8 WIV.Left: Expression in IMR90 KO compared to IMR90 WT .Right: Expression in CS789 compared to CS789 Res .Graphs depict N = 3 biological replicates, mean ± SEM.D After 6 WIV IMR90 KO and CS789 BrainSpheres were treated with 250 nM TubA or 50 nM SAHA for two weeks, while remaining in the orbital shaking incubator.The graphs show marker gene expression at 8 WIV as assessed by qPCR.All graphs depict N = 3 and mean ± SEM.Marker gene expression is normalized to β-actin and respective expression in IMR90 WT .E Illustration of the presumed effect of CSB-deficiency on HDAC-dependent oligodendrocyte maturation.A p-value below 0.05 was termed significant.Statistical analyses were performed using unpaired two-tailed t-tests.Abbreviations: NPCs neural progenitor cells, WIV weeks in vitro, ICC Immunocytochemistry, TubA Tubastatin A. Figure created with biorender.com◂ glia mixed cultures were published by Vessoni et al. [32].Differences in activity might be explained by dissimilarity of patient and control cells' genetic backgrounds, endpoint measures only at one time point, general low network activity or other aspects of the MEA protocol.Part of these limitations could be circumvented by the addition of isogenic control cell lines in our study together with a more robust baseline electrical activity.Treatment with Tubastatin A during the 7 WIV did not antagonize the elevated electrical activity suggesting a different, yet unknown, pathophysiological mode-of-action.
A third neuropathological phenotype found in CSB patients is hypomyelination [1,7,14,51,69].Postnatal lack of myelin in offspring can be evoked by multiple causes ranging from disturbed oligodendrocyte precursor (OPC) cell proliferation, e.g. through Notch pathway inhibition [70], OPC death e.g. by increased oxidative stress or excitotoxicity [71] to inhibited oligodendrocyte maturation, e.g. by deficiency of thyroid hormone [72].Studying hypomyelination in human in vitro models has just recently become possible with appropriate protocols becoming available [44,[73][74][75].We employed hiPSCbased 3D BrainSpheres, which consist of neurons, astrocytes and oligodendrocytes [44] to investigate the hypomyelination phenotype of CSB-deficient BrainSpheres in vitro.While the quantitative ICC analyses showed no difference in the number of O4 + oligodendrocytes in both CSB-proficient and -deficient BrainSpheres, gene expression analyses revealed that CSB-deficient oligodendrocytes do not mature at the same pace as their respective isogenic controls.Similar to NPC migration, also the inhibited oligodendrocyte maturation can be partially rescued through HDAC-inhibition via the HDAC6-specific Tubastatin A and the pan-inhibitor SAHA.A number of studies have suggested a role of HDACs in the regulation of rodent oligodendrocyte differentiation and maturation, which renders them promising targets in different neurological pathologies [76][77][78].Here we show for the first time that altered human oligodendrocyte maturation in an organotypic CSB disease model can be rescued by pharmacological intervention using HDAC inhibitors.
To date, clinically approved HDAC-inhibitors, such as SAHA (aka Vorinostat), are mainly employed as anticancer agents [79,80].Newer developments however, have brought attention to HDAC-inhibitors in other applications, for instance to treat HIV infections, muscular dystrophies, inflammatory diseases, as well as neurodegenerative diseases, such as Alzheimer's Disease, frontotemporal dementia and Friedreich's ataxia [81].HDACs might therefore also be promising targets for developing treatment strategies for CSB.Although the ideal time point of a potential treatment remains to be investigated, an early intervention during child development appears beneficial to prevent disease progression or even weaken the initial development.However, our data suggests that a combination of drugs might be necessary to target the different cellular adversities observed in the CSB-deficient neural models.

Limitations of the study
In our study we used fit-for-purpose hiPSC-derived 3D in vitro models to discover and investigate multiple neurodevelopmental endophenotypes caused by CSB-deficiency.Thereby, we broadly covered multiple cellular pathomechanisms on the expense of deeply uncovering single molecular mechanisms in full detail.For example, we provided first evidence for migration defects correlated with cytoskeletal alterations such as thickened actin stress fibers.Future research should focus on lamellipodia and filopodia which will enhance our understanding of impaired actin migratory structures in CSB patients.Nevertheless, we were able to provide a first line of evidence that HDAC-dependent and -independent mechanisms converge on the pathophysiology of CSB patients.Especially the delayed GABA switch in CSB-deficient hiPSC-based neural in vitro models should be investigated in more detail, as was previously published for Rett Syndrome [82] and schizophrenia [83].Another limitation of our study is the lack of microglia in the neural 3D models.Although not originating from neural stem cells, microglia colonize the developing brain between gestational weeks 4 and 24 [84].They influence brain development by refining CNS formation and function, e.g.synapse formation, circuit sculpting, myelination, plasticity, and cognition.Microglia functional alterations have been associated with neurodevelopmental diseases [85,86].Therefore, especially for testing possible therapeutics in 3D neural models like BrainSpheres, microglia presence will further enhance the predictive value of the models.
With the different neural 3D in vitro models, we provide human-relevant multicellular systems that are already advantageous over tumor cell lines or pure neuronal cultures.However, these structures lack vascularization and, as elaborated above, immune cells.Therefore, these models have limitations when it comes to disease modelling involving inter-organ-crosstalk.Nevertheless, our study shows, that 3D in vitro brain models can provide what animal models often cannot: revealing fundamental disease mechanisms and therapeutic targets.With our work, we aim to spark further investigation into the pathophysiological mechanisms of CSB specifically, and increased usage of hiPSC-based 3D cultures for disease modelling and personalized medicine in general.
Fig.1Human iPSC-derived cell models to study adversities in neurodevelopmental key events linked to Cockayne Syndrome B (CSB).A The two hiPSC-based CSB disease models used in this study include the commercially available IMR90 WT cell line and its CSBdeficient control line IMR90 KO , as well as the patient-derived cell line CS789 and its isogenic control CS789 Res , with partially restored CSB expression.Both IMR90 KO and CS789 Res were produced via CRISPR Cas (see also Supplemental Information Fig.S1).All hiPSC lines were quality controlled and cell banks were prepared according to Tigges et al.[43].B Illustration depicting different fit-for-purpose models.Human iPSCs were neurally induced to hiNPC neurospheres, which were used to assess NPC proliferation after.HiNPCs were plated onto coated surfaces for subsequent differentiation, to assess NPC migration and differentiated into neurons and astrocytes after 3 DIV (middle).In order to obtain electrically active neural networks, hiNPC neurospheres were plated onto coated MEA plates for subsequent differentiation and electrical activity measurement over 7 WIV (left).The differentiation of oligodendrocytes requires a long-term differentiation protocol.HiNPC neurospheres were differentiated in a shaking incubator for 8 WIV to obtain BrainSpheres, which consist of neurons, astrocytes and oligodendrocytes (right).C Immunoblotting of CSB levels in neurospheres of the four hiNPC lines, normalized to the loading control HSP90 and to the respective control (left) or IMR90 WT (right).Representative images are depicted and graphs visualize differences in protein content due to mutations in the CSB gene or between CSB expressing lines, respectively.Graphs depict N = 3 biological replicates, mean ± SEM.Statistical analyses were performed using unpaired two-tailed t-tests.A p-value below 0.05 was termed significant.D HiNPC spheres were plated, differentiated for 3DIV and subsequently stained for nuclei (Hoechst, blue), neurons (MAP2, yellow) and astrocytes (AQP4, magenta).E Transcriptome analyses were performed by plating hiNPC neurospheres onto a coated surface, and differentiating these for 3, 14 and 21 DIV.VENN Diagram of the significantly DEGs between IMR90 WT and IMR90 KO after 3, 14 and 21 DIV, as identified via RNAseq.DEG criteria: |log2| > = 1 and Qvalue of < = 0.05.F 893 DEGs constantly regulated across the differentiation timepoints were mapped to KEGG pathways and the 30 pathways with the highest percentage of regulated genes are depicted.Abbreviations: hiPSC human induced pluripotent stem cells, hiNPCs human induced neural progenitor cells, MEA microelectrode array (MEA), WIV weeks in vitro, DIV days in vitro, DEG differentially expressed gene, RNAseq RNA Sequencing ◂

Fig. 5
Fig. 5 Elevated GABA/Glutamate ratios and delayed KCC2 induction hint towards a delayed GABA switch in CSB-deficient cells.A HiNPC neurospheres were plated and differentiated for 14 DIV, before GC-MS was performed.Graphs depict the ratios of the relative metabolite concentrations of neurotransmitters glutamate and GABA (N = 3 biological replicates, mean ± SEM).B Left: Differential expression of NKCC1 and KCC2 in IMR90 WT and IMR90 KO was evaluated from RNA sequencing data.Graphs depict the FPKM raw values over 21 DIV.Right: NKCC1 and KCC2 mRNA expression in CS789 Res and CS789 was analyzed via qPCR.Graphs depict the FGE